Advances in the areas of the use of recombinant viral vectors for gene therapy and DNA vaccination applications have created a need for large scale manufacture and purification of clinical-grade virus. One such family of viruses are the adenoviruses. The adenoviruses are grouped within the family Adenoviridae, which are split into the genus Aviadenovirus (birds) and Mastadenovirus (human, simian, bovine, equine, porcine, ovine, canine and opossum). A review of the family Adenoviridae can be found in Fundamental Biology, 3rd Ed., Fields, B. N., Knipe, D. M., and Howley, P. M., Ed., at Chapter 30, pp. 979-1016 (1996), which is hereby incorporated by reference. Of specific interest in gene vaccination and/or gene therapy applications is the use of a first generation (FG) replication incompetent adenovirus, crippled by E1 and/or E1/E3 gene deletions, based on serotype 5 of adenovirus. Adenovirus has a broad cell tropism including professional antigen presenting cells such as macrophages and dendritic cells, can infect (if not replicate in) cells from most animal species, and can be produced in large quantities in appropriate human cell lines designed to provide the E1 gene product in trans. The adenovirus genome is generally associated with benign pathologies in humans, and the genomic organization of the virus has been well studied since its discovery in the early 1950s. In addition, the genome is amenable to manipulation, depending on the strategy utilized to construct the respective vector. A replication-incompetent virus (such as an E1/E3 deleted Ad5gag vector expressing a HIV gag transgene, as exemplified herein) requires a cell line which complements the deletions. Any such cell line may be used to generate recombinant virus vectors, with preferred, but not limiting, cell lines including 293 cells and PER.C6™ cells. To this end, numerous 1st generation recombinant adenovirus vectors have been described in the literature (e.g., see Bett, et al., 1994, Proc. Natl. Acad. Sci. 91:8802-8806; WO 01/02607 and WO 02/22080). “Gutless” adenoviral vectors are a 2nd generation adenoviral vector generally devoid of viral protein-coding sequences, frequently with viral proteins supplemented in trans by a helper virus (often an E1-deleted adenovirus) grown with the helper-dependent (HD) adenovector in a packaging cell line (e.g., PER.C6™). Absent viral proteins, these viral vectors can, in the alternative, be supplemented in trans by a cell line and/or “helper virus” capable of expressing the structural and functional adenoviral proteins necessary for successful replication, packaging and rescue. In view of the increased popularity of these viral vectors and the ultimate need to prepare commercial scale quantities of either a viral based vaccine or gene therapy vehicle, it has become essential to devise economical and scalable methods of production and purification.
Early reports of small scale chromatographic purification of adenovirus were reported in the late 1950s and early 1960s (e.g., see Klemperer and Pereira 1959, Virology 9: 536-545; Philipson, 1960, Virology 10: 459-465; Haruna, et al., 1961, Virology: 13 264-267), but was replaced by centrifugation in a CsCl gradient. In the last decade chromatographic purification of adenovirus has again been reported.
U.S. Pat. No. 5,837,520 (see also Huyghe et al., 1995, Human Gene Therapy 6: 1403-1416) disclose a method of purifying adenovirus which comprises treating the cell lysate with a nuclease, followed by (1) anion exchange and (2) metal ion chromatography.
U.S. Pat. No. 6,261,823 discloses a method of purifying adenovirus which comprises subjecting a virus preparation to anion exchange chromatography followed by size exclusion chromatography.
U.S. Pat. No. 6,194,191 discloses methods of purifying adenovirus using low perfusion rates during cell culture, a detergent lysis step, and/or a single chromatography step.
Shabram et al., 1997 (Human Gene Therapy 8: 453-465) discloses a method for measuring Ad5 concentration with analytical anion exchange chromatography.
Despite these reports and others, there remains a need for the development of a large scale process for purification of viral vectors generated within host cell culture systems which address both quantitative and qualitative issues that are imposed upon a commercialized vaccine or gene therapy product. The present invention addresses and meets these needs by disclosing a purification process which, in part, relies upon a selective precipitation step which facilitates removal of vast quantities of contaminating/impure DNA by clarification.